Since the first pregnancy from frozen human mature oocytes (Chen, Pregnancy after human oocyte cryopreservation. Lancet 1986; i: 884-6), various studies have been performed to develop methods for cryopreservation of human oocytes. Surplus mature oocytes from the patients who underwent in vitro fertilization-embryo transfer (IVF-ET) were stored for future use by slow cooling or vitrification methods in a lot of clinics. When the patients failed to become pregnant in their fresh IVF-ET cycles, stored oocytes were recovered and provided for an additional IVF-ET.
Although cryopreservation of human oocytes has performed successfully and also has been introduced widely into human assisted reproductive technology (ART), the clinical outcomes are still limited, because of low pregnancy and implantation rates due to poor viability of thawed oocytes. To improve the viability and quality of oocytes after thawing, modified protocols in slow cooling have been suggested to improve survival rates, e.g., changes involving increase in sucrose concentration (Fabbri et al., Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16:411-6) or the replacement of sodium with choline in the freezing media (Stachecki et al., Detrimental effect of sodium during mouse oocyte cryopreservation. Hum Reprod 1998a; 59: 395-4001998; Quintans et al., Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 2002; 17: 3149-52; Boldt et al, Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum Reprod 2003; 18: 1250-5).
The present inventors have developed a vitrification method for the cryopreservation of human oocytes and got quite a good clinical result (Hong et al., Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil Steril 1999; 72: 142-6; Chung et al., In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil Steril 2000; 73: 545-51; Yoon et al., Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000; 74: 180-1; Yoon et al., Live birth after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2003; 79: 1323-6).
Recently, Martino, et al (Biology of Reproduction, vol. 54, pp 1059-1069, 1996) discloses a method for cryopreserving bovine oocytes in which oocytes were placed in a cryopreservative medium, and placed either in straws or on electron microscope grids. The straws were plunged into liquid nitrogen and the grids were either plunged into liquid nitrogen or nitrogen slush. Martino, et al. report that cooling rates achieved with grids were much higher than with straws. To test whether the use of even faster cooling rates would improve survival, nitrogen slush was compared to liquid nitrogen for freezing oocytes on grids. Survival rates based on morphology, cleavage and blastocyst formation were higher for bovine oocytes frozen in liquid nitrogen compared to those frozen in nitrogen slush.